pilot study: tracer weaner trial for ovine johne’s disease

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Animal Health and Welfare

Pilot Study: Tracer Weaner Trial for Ovine Johne’s Disease National Ovine Johne’s Disease Control and Evaluation Program

Project number TR.073 Final Report prepared for MLA by: NSW Agriculture Elizabeth Macarthur Agricultural Institute PMB 8 Camden NSW 2570 Meat and Livestock Australia Ltd Locked Bag 991 North Sydney NSW 2059 ISBN 1 74036 386 8 JUNE 2000

MLA © 2004 ABN 39 081 678 364

MLA makes no representation as to the accuracy of any information or advice contained in this document and excludes all liability, whether in contract, tort (including negligence or breach of statutory duty) or otherwise as a result of reliance by any person on such information or advice.

Pilot Study: Tracer Weaner Trial for Ovine Johne’s Disease

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ABSTRACT Eradication of ovine Johne’s disease requires destocking sheep and spelling land until Mycobacterium avium subsp. paratuberculoisis has died out. There is no simple way to assess the residual level of contamination or the risk this poses to livestock. Consequently small groups of Merino weaners were orally dosed with M. avium subsp. paratuberculosis. Within 7-14 weeks the organism could be cultured from various locations in the gastrointestinal tract and associated lymph nodes. In addition, the results of a skin test were positive in 66% of weaners in which the organism had established an infection and were negative in uninfected sheep. The results of tests for gamma interferon were positive in some infected weaners but also in some of the controls. An ELISA test was not useful at this early stage of infection. Overall, the results suggest that weaner sheep could be used as sentinels in an infected environment, but this requires validation in a controlled field trial. Furthermore, the results suggest that the infectious dose of M. avium subsp. paratuberculosis may be higher than previously thought.


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EXECUTIVE SUMMARY Eradication of ovine Johne’s disease requires destocking sheep and spelling land until Mycobacterium avium subsp. paratuberculoisis has died out. At the conclusion of a decontamination period, currently deemed to be 2 consecutive summers or 15 months, there is no simple way to assess either the residual level of contamination of the environment or the risk residual contamination poses to livestock. This study was undertaken to determine whether it is possible to detect infection in young sheep exposed to low levels of contamination. Previous experiments in the United Kingdom suggested that this might be possible, but the trials had been done with cattle strains of M. avium subsp. paratuberculosis. Consequently in this project small groups of Merino weaners were orally dosed on repeated occasions with graded numbers of an Australian sheep strain of M. avium subsp. paratuberculosis. Samples were collected from each weaner at regular intervals and evaluated using several tests. Within 7 to14 weeks the organism could be cultured from various locations in the gastrointestinal tract and associated lymph nodes of each sheep that was given a total dose of 1.3 to 6.9 x 107 organisms. Infection was not established in weaners given lower doses. The next lower dose was 8.2 x 103 . Therefore the infectious dose of this strain of M. avium subsp. paratuberculosis is somewhere between about 103 and 107 organisms, a value higher than that found in the UK experiments. Reevaluation of the methods used in the UK experiments suggest that the doses given may have been underestimated by a wide margin. Thus the present results suggest that the infectious dose of M. avium subsp. paratuberculosis may be higher than previously thought with the implication that it may be possible to reduce decontamination periods for land. In other words it might be easier to achieve decontamination of land than previously imagined. Although infection was found by culture of tissues of these weaners, there would be a need to culture all sentinel sheep unless an indirect method could be found to identify sheep most likely to be culture positive. Fortunately the results of a skin test were positive in 66% of weaners in which the organism had established an infection and were negative in uninfected sheep, regardless of whether they had been dosed with the organism or were in a control group. The results of tests for gamma interferon were positive in some infected weaners but also in some of the controls so this test was not of value. An ELISA test also was not useful at this early stage of infection. Overall, the results suggest that weaner sheep could be used as sentinels in an infected environment, and that the skin test could be used to select individual sentinel sheep for culture. This approach requires validation in a controlled field trial. The experimental infection method developed in this study can be used later for other purposes such as evaluating the pathogenesis of ovine Johne’s disease, the behaviour of new diagnostic tests and vaccine efficacy.


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CONTENTS

ABSTRACT................................................................................2

EXECUTIVE SUMMARY ...........................................................3

1. MAIN RESEARCH REPORT ........................................6

1.1 Background and industry context..........................................6

1.2 Project Objectives ...................................................................6

1.3 Introduction..............................................................................6

1.4 Methods ....................................................................................7

1.4.1 Animals.....................................................................................7

1.4.2 Inoculae ....................................................................................7

1.4.3 Clinical and necropsy sampling.............................................8

1.4.4 Serology for antibodies to M avium subsp. paratuberculosis......................................................................8

1.4.5 Gamma interferon assay.........................................................8

1.4.6 Interadermal testing for delayed type hypersensitivity........9

1.4.7 M. avium subsp. Paratuberculosis isolation .........................9

1.4.8 Histopathology.........................................................................9

1.5 Results....................................................................................10

1.5.1 Quantification of M. avium subsp. Paratuberculosis dose 10

1.5.2 Serology for antibodies to M avium subsp. paratuberculosis....................................................................10

1.5.3 Gamma interferon assay.......................................................10

1.5.4 Intradermal testing for delayed hypersensivity ..................12


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1.5.5 Faecal culture.........................................................................13

1.5.6 Gross pathology ....................................................................13

1.5.7 Histopathology.......................................................................13

1.5.8 Culture of tissues for M. avium subsp. paratuberculosis..13

1.6 Discussion..............................................................................14

1.7 Success in achieving objectives..........................................16

1.8 Impact on Meat and Livestock Industry...............................16

1.9 Conclusion and recommendations ......................................17

2. REFERENCES ............................................................17

3. APPENDICES .............................................................20


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1. MAIN RESEARCH REPORT 1.1 Background and industry context Each of the livestock industries in Australia has addressed or is addressing a control program for Johne’s disease. The reason for this is the need to reduce actual or perceived impacts from this disease, which include production losses and trade restrictions, nationally and internationally. Eradication of ovine Johne’s disease requires destocking sheep and spelling land until Mycobacterium avium subsp. paratuberculoisis has died out. At the conclusion of a decontamination period, currently deemed to be 2 consecutive summers or 15 months, there is no simple way to assess either the residual level of contamination of the environment or the risk residual contamination poses to livestock. Work is underway to measure the duration of survival of the organism in soil in experimental plots but these studies will not determine whether infectious doses of the organism remain or whether these viable organisms are available to sheep. For example they might be caught in the soil profile and might pose no risk. For the above reasons it was thought necessary to develop an infection model that uses live sheep. Such an approach is used in other disease control or monitoring programs, particularly for viral diseases, and the animals involved are known as tracers or sentinel livestock. The tracers or sentinels provide an early warning that an infectious disease is present or active. Johne’s disease differs from most other infectious diseases of farm livestock because the incubation period, or time elapsing between infection and signs of sickness, is measured in years not days or weeks. This is a major disadvantage for use of tracer animals. There is a need to find a way of detecting the infection soon after it has occurred. The tracer weaner pilot study described in this report was undertaken for this reason. 1.2 Project Objectives To show that Australian ovine M. avium subsp. paratuberculosis isolates are capable of colonising the intestines of newly weaned merino lambs after oral dosing at levels likely to be representative of those in the field, and that M. avium subsp. paratuberculosis can be detected in these lambs four to six weeks after dosing. To develop an experimental system for M. avium subsp. paratuberculosis infection, providing a tool for further work on pathogenesis and early diagnostic tests. 1.3 Introduction Work by Brotherston et al in the UK in the 1960’s using Cheviot sheep as an experimental model for bovine Johnes disease demonstrated that single or multiple oral doses from 103 to 109 viable Mycobacterium avium subsp. paratuberculosis) could produce detectable infection in intestines and mesenteric lymph nodes of most inoculated sheep as early as four weeks after the last dose. Significantly, this was many months to years before the likely development of clinical disease. We sought to investigate whether this early detection of M.


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avium subsp. paratuberculosis could be utilised to determine the infectivity of pasture following removal of infected sheep, using “tracer-weaner” lambs as “vacuum cleaners” to pick up organisms from pasture. Our study was a pilot pen trial under controlled conditions. In particular we sought to demonstrate that Australian ovine M. avium subsp. paratuberculosis isolates are capable of colonising the intestines of recently weaned merino lambs after oral dosing at levels likely to be encountered during natural infection, and that the organism can be detected in these lambs four to six weeks later. This work would also provide an experimental system for M. avium subsp. paratuberculosis infection, facilitating further work on pathogenesis, early diagnostic tests and vaccines (Trials 1.6 - 1.7). 1.4 Methods 1.4.1 Animals 40 Merino lambs (12-16 wk old) from the EMAI flock (tested negative for Johne’s disease by serology and faecal culture) were weaned onto wire in the EMAI medium security animal house, drenched for internal parasites with Ivomec and Seponver and fed sheep pellets (mainly lucerne in composition) ad-lib. One week before inoculation (2/2/99) they were allocated randomly into groups as detailed in Table 1. All groups were held separately; different dose groups were housed in different rooms, and groups of sheep with different schedules of the same dose were separated by double weldmesh partitions in the same room. Any intervention (eg routine cleaning, sampling or dosing) was done in order: control then low then medium then high dose groups. Disinfectant footbaths were used on exit from each room, operators wore disposable plastic coveralls and all effluent was heat-treated at 70 oC for one hour. Lambs were euthanased with IV barbiturate 5 weeks after the last dose. 1.4.2 Inoculae M. avium subsp. paratuberculosis was isolated from six separate faecal samples collected from sheep from a known infected farm in modified Bactec medium 1, sub-cultured onto slopes of modified Middlebrook 7H10 medium and its identity verified by IS900 and IS1311 PCR with REA 2 3. Colonies were harvested into phosphate buffered saline (PBS) from these slopes, suspended, pooled and used to inoculate another Bactec vial which was used as stock culture. Subcultures into fresh Bactec vials were made from this vial when required. These were then subcultured after 3-4 weeks onto modified 7H10 slopes. Colonies from six 6-week-old slopes with confluent growth were harvested into a total volume of 1ml PBS. This was termed “neat suspension”, some of which was used immediately, and the remainder stored at 4 oC until used. Three separate neat suspension preparations were used, one for inoculations at weeks 0, 1 and 2, the second for weeks 3, 4 and 5, and the third for weeks 6 to 9. The total number of M. avium subsp. paratuberculosis (viable and non-viable) in the suspensions was determined by direct counts of appropriate dilutions in a Thoma counting chamber. Based on previous experiments the numbers of viable M. avium subsp. paratuberculosis were estimated to be 0.5 to 1 log lower than the direct count. Neat suspension was diluted in PBS immediately before each inoculation to provide the intended dose for each lamb (see Table 1) in a volume of 2 ml, which was delivered to the back of the pharynx using a syringe with bulb-ended needle. The actual dose in terms of viable organisms was determined retrospectively from the neat suspensions by culture in modified Bactec media using replicate serial dilutions to provide most probable number (MPN) estimates. Briefly, for each MPN estimate five 0.1 ml replicates of each of seven serial tenfold dilutions were inoculated into Bactec vials, and growth indices measured weekly. The


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MPN estimates were read from published tables 4 using the culture results (number of positive samples from the five replicate vials) for three appropriate sequential dilutions. Because there may be loss of viability of M. avium subsp. paratuberculosis during storage at 4 oC, viable estimates were repeated at weeks 2, 5 and 9 for each suspension respectively and the average number of viable organisms in each dose was determined. Thus an MPN estimate was made for each time point when inoculation of lambs was conducted. 1.4.3 Clinical and necropsy sampling Lambs were sampled immediately before the first dose, fortnightly thereafter and on the day of necropsy (see Table 1). One faecal pellet was collected manually from the rectum of each lamb at each sampling time. Blood was collected from the jugular vein into plain and lithium heparin vacutainers. In addition, faecal samples were collected daily from the high and medium dose groups in the extended dose regime. At necropsy a range of tissues (caecum, colon, , ileocaecal valve, terminal ileum x 4, jejunum - with and without Peyers patches, duodenum, ileocaecal node, caudal mesenteric node, two more mesenteric nodes, retropharangeal node and pharangeal tonsil) were collected for M. avium subsp. paratuberculosis isolation. The same range of tissues was also collected into 10% neutral buffered formalin for histopathology. In addition duplicate samples were collected from the above range of tissues, plus a further 35 sites, and stored at -80 oC, pending further investigation (Appendix 6). 1.4.4 Serology for antibodies to M avium subsp. paratuberculosis Plain blood samples were allowed to clot and retract, and serum was removed and stored at -20 oC for up to 4 months until assayed using a commercial kit (PARACHEK Johne’s absorbed EIA, CSL, Parkville, Victoria) according to manufacturer’s instructions. 1.4.5 Gamma interferon assay Lithium heparin blood samples were assayed for gamma interferon (IFN-γ) using a commercial test kit (Bovigam, Bovine gamma interferon test, CSL, Parkville, Victoria) according to manufacturer’s recommendations but with slight modifications. The kit was developed for use with cattle. Briefly, three 1.5 ml aliquots of well mixed heparinised blood were incubated for 18 hours at 37 oC in eppendorf tubes with 100 µl of bovine PPD, avian PPD (both 300 µl/ml) and PBS, respectively. Plasma was collected after centrifugation at 500 g for 10 minutes, and stored frozen at -20 oC for up to five months. Samples were thawed overnight at 4 oC, allowed to equilibrate to room temperature and thoroughly mixed by vortexing before use in the enzyme immunoassay (EIA). The EIA was performed exactly according to the manufacturer’s recommendations. Briefly, samples were added to 96 well microtitre plates coated with antibody to bovine IFN-γ, incubated and washed. Conjugate (horseradish peroxidase labelled anti-bovine IFN-γ) was added, incubated and washed. Enzyme substrate was added, incubated, the reaction was stopped and absorbance was read at 450 nm. Sera were again frozen at -20 oC for a further 4 months and the EIA was the repeated after centrifugation of the thawed plasma samples to remove any clots which had formed.


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1.4.6 Interadermal testing for delayed type hypersensitivity One week before euthanasia lambs were injected intradermally on the wool-free inner thigh/inguinal region with 0.1 ml Johnin PPD (0.5 mg/mL, CSL, Parkville, Victoria). Skin thickness was measured with vernier callipers before injection and 72 hours later. 1.4.7 M. avium subsp. Paratuberculosis isolation The samples collected fortnightly and at necropsy were processed on the day of collection. Additional daily faecal samples were frozen at -80 oC for up to 5 months until processed. Faecal samples were prepared by the double incubation method of Whitlock and Rosenberger 5. Briefly, the faecal pellet was placed in a 15 ml polypropylene tube and broken up in 10-12 ml of sterile normal saline using a swab stick. After mixing, the tube was allowed to stand for 30 min at room temperature. A 5ml aliquot of the surface fluid was transferred to a 35ml polystrene tube containing 25ml of 0.9% hexadecylpyridinium chloride (HPC) (Sigma Chemical Co., St Louis, Mo) in half-strength brain heart infusion broth (Oxoid, Basingstoke, England) and allowed to stand at 37 oC for 24 h. The tube was centrifuged at 900 g for 30 mins. The pellet was resuspended in 1 ml sterile water with vancomycin (100 µg/ml), nalidixic acid (100 µg/ml) and amphoteracin B (50 µg/ml) (all Sigma) and incubated for 48 to 72 h at 37 oC. 0.1ml was then inoculated into Bactec vials. Tissue samples (approximately 1-5 g) were trimmed of fat and fibrous tissue and homogenised for 30 s in 2 ml sterile normal saline in a blender. After adding 25 ml 0.75% HPC, the homogenates were left standing at room temperature for 72 h and 0.1 ml of sediment was collected carefully from the bottom of the tube for inoculation into Bactec vials. The modified Bactec 12B radiometric medium consisted of 4 ml enriched Middlebrook 7H9 medium (Bactec 12B; Becton Dickinson, Sparks, Md.) with 200 µl PANTA PLUS (Becton Dickinson), 1 ml egg yolk, 5 µg mycobactin J (Allied Monitor Inc., Fayette, Mo.) and 0.7 ml water 1. The vials were incubated at 37 oC for up to 12 weeks and growth indices were measured weekly with an automatic ion chamber (BACTEC 460; Johnston Laboratories, Towson, Md). Samples yielding a positive growth index were then assayed by PCR with REA1. 1.4.8 Histopathology Tissues (above) were processed routinely for histopathology, sectioned at 5 µm, stained with haematoxylin & eosin (H&E) and a Ziehl-Neelsen technique (ZN), and examined by light microscopy. Table 1: Experimental design Group No. of

lambs M ptb per dose (intended)

Freq of dosing

Total dose (intended)

Clinical sampling, week number

Time of necropsy

s: control 5 0 3x1 wk 0 0,2,4,6,7 wk 7 s: low dose 6 102 .. 3x102 .. .. s: medium dose 6 104 .. 3x104 .. .. s: high dose 3 108 .. 3x108 .. .. e: control 5 0 10x1 wk 0 0,2,4,6,8,10,12,14 wk 14 e: low dose 6 102 .. 103 .. .. e: medium dose 6 104 .. 105 .. .. e: high dose 3 108 .. 109 .. .. s = short regime (3 doses one week apart) e = extended regime (10 doses one week apart)


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1.5 Results 1.5.1 Quantification of M. avium subsp. Paratuberculosis dose The direct counts conducted by microscopy and the MPN estimations conducted by culture for each neat suspension used in the preparation of inocula are given in Table 2. MPN estimations for viable M. avium subsp. paratuberculosis were up to 2 logs lower than the direct counts. The actual numbers of viable M. avium subsp. paratuberculosis organisms in each dose were about one log lower than the intended dose (Table 3). Table 2: Direct microscopic counts of M. avium subsp. paratuberculosis compared to MPN estimates of viable organisms in suspensions used to prepare experimental inocula. Suspension Log10 Direct

count Log10 MPN estimation

95% confidence limits for MPN estimation

lower upper Weeks 0,1,2 day prepared at week 0 10.3 8.52 8.04 8.97 at week 2 10.3 8.04 7.49 8.40 Weeks 3,4,5 day prepared at week 3 10.3 9.11 8.54 9.48 at week 5 10.3 7.90 7.40 8.28 Weeks 6,7,8,9 day prepared at week 6 10.2 8.23 7.65 8.69 at week 9 10.3 7.0* * No MPN estimate was possible due to media contamination. The figure given is an estimate based on previous storage experiments in PBS and the other suspensions in this experiment. Table 3: Intended and actual doses of M. avium subsp. paratuberculosis Group M paratb per

dose (intended) M paratb per dose (actual)

Freq of dosing

Total dose (intended)

Total dose (actual)

S: control 0 0 3x1 wk 0 0 S: low dose 102 8.7x100 .. 3x102 2.6x101 S: med dose 104 8.7x102 .. 3x104 2.6x103 S: high dose 108 4.4x106 .. 3x108 1.3x107 E: control 0 0 10x1 wk 0 0 E: low dose 102 8.2x100 .. 103 8.2x101 E: med dose 104 8.2x102 .. 105 8.2x103 E: high dose 108 6.9x106 .. 109 6.9x107 s = short regime (3 doses one week apart) e = extended regime (10 doses one week apart) 1.5.2 Serology for antibodies to M avium subsp. paratuberculosis One sheep (number 40) from the high dose group gave a positive EIA result at week 14, using the recommended interpretation criteria. No positive results were obtained for any other sheep or at any other time during this trial. (See Appendix 3 for full tabulation of results). 1.5.3 Gamma interferon assay In the first run of the EIA, many samples, including unstimulated controls, gave very high optical densities but there was an apparently random pattern. This appeared to be related to plasma clots that formed in the tubes after thawing. The spurious high values were not seen when the assay was repeated after centrifugation of the thawed plasma samples. The corrected optical density values (PPD-stimulated minus nil-antigen) from the latter assay are given in Table 4. (Complete results are given in Appendix 2). The positive-negative cutt-off


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was arbitrarily defined as the mean plus 3 standard deviations of the day 0 values (0.029 + 3 x 0.053 = 0.188). Assuming a normal distribution of test results, 99% of truly negative samples would have an optical density lower than the cut-off. Using the cut-off of 0.188, two sheep (number 8 from the control group and number 28 from the medium dose group) had positive values on day 0 (i.e. before dosing), and these values tended to remain elevated, although not always exceeding the cut-off, in subsequent samplings. Sheep 34 (medium dose) had positive values at every sampling, except day 0 when the value approached the cut-off point. Three other sheep (number 31 from the medium dose groups, and numbers 36 and 38 from the high dosed groups) had positive values on at least one occasion from seven weeks after the first dose. The gamma-interferon responses were compared between groups at each sampling time with a non-parametric test (Mood’s median test, Minitab statistical software). There were significant differences between group medians for weeks 8 and 14 (high > control) (P<0.05).


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1.5.4 Intradermal testing for delayed hypersensivity The skin thickness was measured immediately before and 72 hours after intradermal injection of 0.1 ml Johnin. An increase in thickness of > 0.5 mm was considered a positive reaction. One of three high dose, short regime lambs (number 36) was positive at 6 weeks

Table 4. Gamma-interferon results, Avian PPD minus nil-antigen

W eek of sampling after first doseSheep 0 2 4 6 7 8 10 12 14

Undosed controls 1 0.000 0.018 0.018 0.020 0.0052 0.000 0.019 0.005 0.006 0.0283 -0.015 0.000 0.000 -0.009 0.0087 0.004 0.006 0.019 0.016 0.0858 0.205 0.053 0.088 0.059 0.0484 -0.001 0.012 -0.006 0.010 -0.002 0.004 0.000 -0.0065 0.022 0.031 0.029 0.022 0.001 0.026 0.014 0.0126 0.040 -0.051 0.065 0.048 -0.001 -0.042 0.003 0.0099 0.006 -0.007 0.002 0.005 0.003 -0.129 0.005 0.030

10 0.154 -0.071 0.094 0.115 0.021 0.107 -0.003 -0.023Means 0.042 0.001 0.031 0.029 0.035 0.004 -0.007 0.004 0.004

Low dose 11 0.007 0.013 0.011 0.004 0.01712 0.007 0.007 -0.066 0.003 0.01713 0.010 0.008 0.005 0.021 0.00714 0.010 -0.001 0.021 0.003 0.05215 0.002 0.004 0.018 0.046 0.07316 0.004 0.038 0.023 0.017 0.01617 0.046 0.016 -0.010 0.006 0.023 0.099 0.038 0.05018 0.003 0.010 -0.002 0.005 0.006 0.006 -0.003 0.01319 0.020 0.010 -0.014 0.010 -0.013 0.024 0.004 0.05520 0.009 -0.004 0.011 0.013 0.016 0.015 0.009 -0.03021 0.007 0.019 0.006 0.007 0.033 0.028 0.003 0.14222 0.004 0.021 0.001 -0.001 0.169 -0.014 -0.003 0.033

Means 0.011 0.012 0.000 0.011 0.030 0.039 0.026 0.008 0.044

M edium dose23 0.015 0.035 0.065 0.023 0.03724 -0.008 -0.005 0.001 0.017 0.02425 0.047 0.008 0.011 -0.001 0.01926 0.020 -0.004 0.008 0.000 0.00127 0.018 -0.006 -0.003 0.001 0.04028 0.203 0.072 0.589 0.028 0.12029 0.008 0.000 0.004 -0.004 0.013 -0.002 -0.002 0.01330 0.051 0.025 0.080 0.020 0.073 0.053 0.004 -0.00731 0.009 0.008 0.012 0.010 0.041 0.003 0.204 0.00232 0.001 0.023 0.029 0.008 0.007 0.049 0.011 0.01233 0.001 0.009 0.031 0.002 0.026 0.116 0.012 -0.04134 0.123 0.260 0.425 0.339 0.555 0.245 0.225 0.269

Means 0.041 0.035 0.104 0.037 0.040 0.119 0.077 0.076 0.041

High dose35 -0.003 0.005 0.005 0.001 0.08436 0.084 0.076 0.120 0.036 0.22837 0.009 -0.002 0.001 -0.007 0.02338 -0.005 0.014 0.011 -0.003 0.230 0.038 0.096 0.30039 0.049 0.028 0.063 0.030 0.104 0.159 0.068 0.03240 0.008 0.006 0.007 0.003 0.039 0.090 0.103 0.052

Means 0.024 0.021 0.035 0.010 0.112 0.124 0.096 0.089 0.128* *

Positive samples positive using cut-off point 0.188 (Day 0 mean + 3 standard deviations)* Medians for these groups exceed controls (P< 0.05, Moon's m edian test)


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after the first dose, and all three high dose, long regime lambs (numbers 38,39,40) were positive 13 weeks after the first dose. One low dose, long regime sheep (number 20) had an equivocal reaction (thickness increase 0.5 mm) at week 13 and all other sheep were negative. Using tissue culture (see below) as the gold standard test, the sensitivity of skin testing was 66% overall (100% for the lambs necropsied at week 14) and the specificity was 100%. (Detailed results for each lamb are given in Appendix 1). 1.5.5 Faecal culture All cultures from faecal samples collected fortnightly were negative suggesting that there was no excretion of M. avium subsp. paratuberculosis during the trial. However, positive results were obtained from some of the faecal samples collected daily. Three sheep in the high dose groups were culture positive on 4 separate occasions between days 1 to 6 after dosing. The latter results are consistent with passive shedding of orally-dosed organisms. (See Appendix 5 for complete results). 1.5.6 Gross pathology No significant gross lesions were present in any sheep. 1.5.7 Histopathology No lesions were present in any tissue in any sheep. ZN stained sections revealed a clump of three acid-fast organisms in a macrophage in a single lymph node from one high dose, short regime lamb (number 35). No other tissue from this or any other sheep had visible acid-fast organisms. 1.5.8 Culture of tissues for M. avium subsp. paratuberculosis One or more tissues from each sheep in the high dose groups were culture positive however, none of the sheep in the control, low or medium dose groups had culture positive tissues (Table 5). The organism was recovered from at least one tissue of all 6 sheep which had received the high dose level, for both short and extended dosing regimes (7 and 14 weeks after first infection). M. avium subsp. paratuberculosis was recovered from the terminal ileum of 4 sheep, the ileocaecal valve of 3 sheep, the mesenteric lymph nodes of 4 sheep, the tonsil of 2 sheep and the jejunum of 1 sheep. The contamination rate for these 600 tissue cultures was zero, i.e. M. avium subsp. paratuberculosis was confirmed by PCR in each culture that developed a growth index. (Complete results are given in Appendix 4).


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1.6 Discussion Infection of weaned Merino lambs with an Australian isolate of an ovine strain of M. avium subsp. paratuberculosis was demonstrated, with total doses from about 107 to 108 viable organisms. A total dose of 104 or fewer organisms did not produce detectable infection in this trial. Thus we have demonstrated that the lowest infectious dose for lambs under these experimental conditions is somewhere between 104 and 107 viable organisms. Apart from a single trial in which Brotherston et al 6 infected lambs with 103 organisms, most previous experimental infections in sheep have used doses in excess of 109, and often greater than 1010 organisms7 8-12. It is important to realise that lambs in this trial were successfully infected with doses ranging from 0.1% to 1% of those used in most of the research trials conducted worldwide to date. With regard to the lowest infectious doses reported by Brotherston et al6 , we were unable to infect lambs with similar levels. However, there were significant differences in the methods of quantification of the organisms between the present trial and that of Brotherston et al6 . Brotherston et al 6 used colony counts on solid media to estimate the number of organisms. Our experience is that under ideal cultural conditions the number of viable M. avium subsp. paratuberculosis obtained by this technique may be at least 10-fold lower than that obtained using liquid Bactec media. Moreover, the organisms used by Brotherston et al6 had cultural characteristics of the bovine strain of M. avium subsp. paratuberculosis. This strain has a pronounced tendency to form clumps when attempts are made to form a suspension, and this results in significant underestimation of the actual count when plate counting methods are used. Thus their low dose was probably in excess 104 and possibly in excess of 105 organisms. Nevertheless, it would appear from the results of the present trial where accurate enumeration techniques were employed that the infectious dose of ovine strain M. avium subsp. paratuberculosis may be somewhat higher than previously imagined. We have previously quantified numbers of M. avium subsp. paratuberculosis in faeces from known infected sheep, and measured levels up to 108 organisms per gram13. Because of the elaborate decontamination procedure required to isolate M. avium subsp. paratuberculosis from faecal material, this measured number of organisms may be about one hundred-fold less than the likely real value14. Thus levels of 1010 organisms per gram of faeces are

Table 5: Culture results, high dose groups

Tissue

Sheep Schedule Col

on

Cae

cum

ICV

Term

inal

ileu

m

Term

inal

ileu

m

Term

inal

ileu

m

Jeju

num

+ P

P

Jeju

num

- PP

Duo

denu

m

Mes

ente

ric L

N

Mes

ente

ric L

N

Mes

ente

ric L

N

Mes

ente

ric L

N

Ret

roph

aran

geal

LN

Tons

il

35 Short - - - - p p - - - p p p - - p36 Short - - p - - - - - - - p - - - -37 Short - - - p - - - - - - - - - - -38 Extended - - - - - - - - - - - - - - p39 Extended - - p p - p - - - p p - - - -40 Extended - - p p p - p p - p p p p - -

p = positive


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possible in sheep with multibacillary paratuberculosis. In the field, ingestion of gram quantities of faeces is quite likely, especially over time, so that lambs in a paddock with infected sheep would certainly be exposed to such levels, which are 100 to 1000-fold above the levels we have demonstrated to be infectious in this trial. Moreover, we infected sheep using cultured organisms, and it is possible that cultured organisms may be less pathogenic than those excreted by infected sheep. In other words, for reasons outlined above we believe sheep in the field are likely to be exposed to levels of contamination as high or higher than those used in this trial, and the infectious dose for sheep exposed to natural faecal material may be lower than that measured in this trial. However, contamination levels on pasture after infected sheep have been removed will drop over time. “Hot spots” in spelled paddocks, such as around sheep camps, watering points, hand feeding areas etc could have high residual levels, even long after average levels of contamination on pasture dropped below an infectious level. The chance of exposure of lambs to infective levels of M. avium subsp. paratuberculosis in the field can only be assessed by extending the laboratory “tracer weaner” model to a controlled paddock trial. The use of the skin test to assess cell mediated immunity to M. avium subsp. paratuberculosis was surprisingly successful. We detected 100% of culture-confirmed infected sheep at the high dose, extended regime, and 66% of culture-confirmed infected sheep overall, with 100% specificity. In cattle, skin testing is rarely used because it has been found repeatedly to have both low sensitivity and low specificity15. However, while most reports suggest that the sensitivity of the skin test is quite low in adult sheep16, the specificity can be high, both in the experimental situation 17 and in the field16. Little attention has been paid to lambs, but early experimental work suggests that skin testing can be quite sensitive soon after infection, with responses declining over time18. It is important to note that the results in our trial were obtained for young lambs in an animal house situation and probably with minimal exposure to environmental mycobacteria which might stimulate cross-reactivity in the skin test. Thus we were able to use a relatively small increase in skin thickness (>0.5mm) as a positive criterion without loss of specificity. For adult sheep in overseas work, an increase in skin thickness of >4mm is used. The simplicity of the skin test would make it ideal for use in the field situation to monitor exposure of tracer lambs, hopefully allowing targeted necropsy and culture to confirm infection status. It should be investigated in a controlled field trial. There was also some evidence for the development of cell-mediated immunity to M. avium subsp. paratuberculosis in the gamma-interferon results of these lambs, the high dose group having significantly higher medians than the control group at weeks 8 and 14. Considering individual sheep, two (numbers 36 and 38) of six high dose animals had elevated values on at least one occasion beyond week 7, and interestingly both of these also had positive results in the skin test. However, three other sheep (numbers 8, 28 and 34) had elevations of similar or greater magnitude that were quite unrelated to any exposure to M. avium subsp. paratuberculosis. This lack of specificity would limit the use of this test in the very early stages of the disease in lambs. Further research with assessment of sheep at later times after infection and with alternative positive-negative cut-off points might be worthwhile. As expected there was little evidence for serological response to M. avium subsp. paratuberculosis in this trial with only one animal showing a positive result at the final sampling.


16

The use of the skin test in this trial, one week before the final sample for other immunologic tests, was also designed to provide an anamnestic stimulus for those tests. We did not see any obvious evidence for this in the results. The single positive result in the final ELISA for serum antibodies in one high dose animal only is suggestive but no conclusions can be drawn. In the gamma-interferon test, 3 sheep had positive results one week after skin testing. Animals 36 (week 7) and 38 (week 14) were positive on the skin test indicating that a CMI response was already present and sheep 34 had consistently shown high levels from repeated samplings. No conclusions can be drawn from these results. Again, further research should be contemplated. Some discussion is warranted regarding the isolation of M. avium subsp. paratuberculosis from the faeces of experimental animals between 1 and 6 days after dosing. This probably represents passive excretion . The timing is as previously reported for passive excretion in cattle 19-22 , an event which was demonstrated to follow the same pattern as inert particulate matter19. Juste et al 23 cultured the organism from faeces of lambs up to 10 days after experimental infection with 150 mg wet wt of M. avium subsp. paratuberculosis (probably about 1010 organisms). In the present trial passive excretion was demonstrated after doses as small as 4.4 x 106 organisms in lambs which had no gross or histological evidence of paratuberculosis, although the organism was later recovered at necropsy from the tissues. Thus an occasional animal in an infected flock will be positive on faecal culture, and itself have little evidence for infection at necropsy. Due to passive excretion the specificity of faecal culture for M. avium subsp. paratuberculosis for the disease status of an individual animal may be less than 100%; if, however it is taken as a measure of infection in the flock, 100% specificity would be expected. It is important to appreciate that the present trial aimed to study only the initial infection of lambs with M. avium subsp. paratuberculosis. Whether individual infected lambs would go on to eliminate the organism, become chronic asymptomatic carriers or clinical cases cannot be assessed from such a short -duration trial. Nonetheless, we did detect infection with viable M. avium subsp. paratuberculosis in sheep tissues, including extra-intestinal tissues, five weeks after the last exposure to the organism, indicating a carrier state at that time. These lambs were certainly at risk of becoming subclinical or clinical cases of Johne's disease, and a potential source of infection to other sheep. However, the ultimate fate of these sheep is not important in the context of the tracer-weaner model. 1.7 Success in achieving objectives Both objectives were achieved. The Australian sheep strain of M. avium subsp. paratuberculosis was shown to be capable of infecting sheep and colonising the intestine of weaners after realistic doses. This colonisation could be detected within a few months of giving the first dose. An experimental system has been developed for M. avium subsp. paratuberculosis infection and this can be used for further work on pathogenesis and early diagnostic tests. 1.8 Impact on Meat and Livestock Industry It is difficult to forecast an immediate or long term impact from this work until a controlled field trial has been undertaken. The concept of infecting weaners and then detecting the infection has worked very well in a pen trial but it is not possible to extrapolate these findings to the real world. In nature doses of the organism may be acquired over a prolonged period, animals would be exposed to a wide variety of environmental bacteria and there may be


17

considerable differences in degree and timing of exposure between farms and geographic regions. Application of the tracer weaner concept in the national program is still some way off. However, there are some direct and useful outcomes from the work completed so far. The collective impacts are: A field trial of the tracer weaner concept is justifiable based on trial results to date. The infectious dose of M. avium subsp. paratuberculosis appears to be higher than previously thought. This finding should be greeted with optimism because it reinforces the concept that spelling land after removing infected sheep is a viable means of ridding a farm of ovine Johne’s disease. It may mean that spelling periods can be reduced. This finding needs to be considered together with the results from separate trials on the duration of survival of the organism in soils. A useful means of infecting weaners under controlled experimental conditions has been developed. It employs relatively low doses of the organism that may reflect natural levels of exposure and it uses small group sizes. Other experimental models in current use employ unrealistically high doses and cause an unnatural kind of infection in which too large a proportion of animals develop too severe an infection too quickly. The results from such trials are unlikely to be representative of field situations. The new infection model may be applicable in other studies of diagnostic tests, vaccines and the pathogenesis of ovine Johne’s disease and is likely to provide a better indication of what can be expected in nature. 1.9 Conclusion and recommendations Infection with M. avium subsp. paratuberculosis was demonstrated at 7 or 14 weeks after first exposure in each of 6 lambs given total oral doses of 1.3 to 6.9 x 107 viable organisms. There were no associated gross or microscopic lesions due to the short duration of this trial. Only one lamb gave a positive result in ELISA for antibodies to M. avium subsp. paratuberculosis. There was some correlation between gamma interferon test outcome and infection status, but this test lacked specificity. Intradermal testing with Johnin PPD enabled ante-mortem detection of all three culture-confirmed infected lambs at 13 weeks, and one of three culture-confirmed infected lambs at 6 weeks after first exposure to M. avium subsp. paratuberculosis with 100% specificity. The results from this trial suggest that the tracer weaner concept should be evaluated in the field. 2. REFERENCES 1. Whittington RJ, Marsh I, McAllister S, Turner MJ, Marshall DJ, Fraser CA.

Evaluation of Modified BACTEC 12B Radiometric Medium and Solid Media for Culture of Mycobacterium avium subsp. paratuberculosis from Sheep. J Clin Microbiol 1999; 37(4):1077-83.

2. Marsh I, Whittington R, Cousins D. PCR-restriction endonuclease analysis for

identification and strain typing of Mycobacterium avium subsp. paratuberculosis and Mycobacterium avium subsp. avium based on polymorphisms in IS 1311. Mol Cell Probes 1999; 13(2):115-26.

3. Whittington RJ, Marsh I, Turner MJ et al. Rapid detection of Mycobacterium

paratuberculosis in clinical samples from ruminants and in spiked environmental


18

samples by modified BACTEC 12B radiometric culture and direct confirmation by IS900 PCR. J Clin Microbiol 1998; 36(3):701-7.

4. Anon. AS 1766.1.6 - Food microbiology, Method 1.6: General procedures and

techniques - estimation of most probable number (MPN) of microorganisms. Australian Standard 1991.

5. Whitlock RH&RAE. Fecal culture protocol for {IMycobacterium paratuberculosis}.

A recommended procedure. In: Proceedings of the Annual Meeting of the United States Animal Health Association. Denver 1990. Pp. 280-5.

6. Brotherston JG, Gilmour NJL, Samuel JMcA. 1 Quantitative studies of

Mycobacterium johnei in the tissues of sheep. 1 Routes of infection and assay of viable M. johnei. J Comp Pathol 1961; 71:286-98.

7. Begara-McGorum I, Wildblood LA, Clarke CJ et al. Early immunopathological

events in experimental ovine paratuberculosis. Vet Immunol Immunopathol 1998; 63. (3.):265-87.

8. Williams ES, Snyder SP, Martin KL. Experimental infection of some North

American wild ruminants and domestic sheep with Mycobacterium paratuberculosis: clinical and bacteriological findings. J Wildl Dis 1983; 19(3):185-91.

9. Allen WM, Berrett S, Patterson DS. A biochemical study of experimental Johne's

disease. II. An in vitro study of L-histidine uptake by sheep intestinal mucosa. J Comp Pathol 1974; 84(3):385-9.

10. Garcia Marin JF, Benazzi S, Perez V, Badiola JJ. Study of the entrance of M.

paratuberculosis in the lambs intestinal mucosa using immunohistochemical methods for antigen detection. 3rd Inter Colloq Paratuberc, Providence, RI, USA 1992; 370-7.

11. Kluge JP, Merkal RS, Monlux WS et al. Experimental paratuberculosis in sheep

after oral, intratracheal, or intravenous inoculation lesions and demonstration of etiologic agent. Am J Vet Res 1968; 29(5):953-62.

12. Stewart D, Vaughan J, Noske P, Jones S, Tizard M, Prowse S. Immunological and

bacterial time course studies on experimental Mycobacterium paratuberculosis infections in sheep, goats and calves. 6th International Colloquium on Paratuberculosis, Melbourne, Australia 1999.

13. Whittington RJ, Reddacliff LA, Marsh I, McAllister S, Saunders V. Temporal

patterns and quantification of excretion of Mycobacterium avium subsp paratuberculosis in sheep with Johne's disease. Aust Vet J 2000; 78(1):34-7.

14. Mokresh AH, Czuprynski CJ, Butler DG. A rabbit model for study of

Mycobacterium paratuberculosis infection. Infect Immun 1989; 57(12):3798-807. 15. Chiodini RJ, Van Kruiningen HJ, Merkal RS. Ruminant paratuberculosis (Johne's

disease): the current status and future prospects. Cornell Vet 1984; 74(3):218-62.


19

16. Perez V, Tellechea J, Corpa JM, Gutierrez M, Garcia Marin JF. Relation between

pathologic findings and cellular immune responses in sheep with naturally acquired paratuberculosis [In Process Citation]. Am J Vet Res 1999; 60(1):123-7.

17. Gilmour NJL, Halhead WA, Brotherson JG. Studies of immunity to Mycobacterium

johnei in sheep. J Comp Pathol 1965; 75:165-73. 18. Gilmour NJL, Angus KW, Mitchell B. Intestinal infection and host response to oral

administration of Mycobacterium johnei in sheep. Veterinary Microbiology. 1978; 2. 223-35.

19. Sweeney RW, Whitlock RH, Hamir AN, Rosenberger AE, Herr SA. Isolation of

Mycobacterium paratuberculosis after oral inoculation in uninfected cattle. Am J Vet Res 1992; 53(8):1312-4.

20. Larsen AB, Merkal RS, Moon HW. Evaluation of a paratuberculosis vaccine given

to calves before infection. Am J Vet Res 1974; 35(3):367-9. 21. Larsen AB, Merkal RS, Cutlip RC. Age of cattle as related to resistance to infection

with Mycobacterium paratuberculosis. Am J Vet Res 1975; 36(3):255-7. 22. McDonald WL, Ridge SE, Hope AF, Condron RJ. Evaluation of diagnostic tests for

Johne's disease in young cattle . Aust Vet J 1999; 77(2):113-9. 23. Juste RA, Garcia Marin JF, Peris B, Saez de Ocariz CS, Badiola JJ. Experimental

infection of vaccinated and non-vaccinated lambs with Mycobacterium paratuberculosis. J Comp Pathol 1994; 110(2):185-94.


20

3. APPENDICES Appendix 1: Skin test results Sheep Schedule Dose Skin thickness, 0 hr Skin thickness, 72 hr Increase Swelling

Meas

1 Meas

2 Average Meas

1 Meas

2 Average 1 Short Control 0.8 1.0 0.90 0.7 0.8 0.75 -0.15 - 2 Short Control 1.2 1.0 1.10 1.1 1.0 1.05 -0.05 - 3 Short Control 1.0 0.8 0.90 0.7 1.0 0.85 -0.05 - 4 Extended Control 0.6 0.7 0.65 1.1 0.9 1.00 0.35 - 5 Extended Control 0.7 0.8 0.75 0.9 0.7 0.80 0.05 - 6 Extended Control 0.8 0.6 0.70 0.6 0.7 0.65 -0.05 - 7 Short Control 1.0 1.0 1.00 1.0 0.8 0.90 -0.10 - 8 Short Control 1.1 1.0 1.05 1.0 1.0 1.00 -0.05 - 9 Extended Control 0.7 0.6 0.65 0.9 1.0 0.95 0.30 - 10 Extended Control 0.8 0.9 0.85 0.7 0.6 0.65 -0.20 - 11 Short Low 1.0 0.9 0.95 0.9 0.6 0.75 -0.20 - 12 Short Low 0.8 0.8 0.80 1.0 0.9 0.95 0.15 - 13 Short Low 0.8 1.0 0.90 1.0 1.1 1.05 0.15 - 14 Short Low 1.0 1.1 1.05 1.0 0.8 0.90 -0.15 - 15 Short Low 0.9 0.9 0.90 1.0 1.0 1.00 0.10 - 16 Short Low 0.8 0.8 0.80 0.9 1.0 0.95 0.15 - 17 Extended Low 0.6 0.6 0.60 0.8 0.8 0.80 0.20 - 18 Extended Low 0.8 0.7 0.75 0.7 0.9 0.80 0.05 - 19 Extended Low 0.7 0.7 0.70 1.0 0.8 0.90 0.20 - 20 Extended Low 0.6 0.6 0.60 1.0 1.2 1.10 0.50 10* 21 Extended Low 0.9 0.9 0.90 1.0 0.6 0.80 -0.10 - 22 Extended Low 0.8 0.6 0.70 0.8 0.8 0.80 0.10 - 23 Short Medium 0.8 0.9 0.85 0.8 0.9 0.85 0.00 - 24 Short Medium 1.1 0.9 1.00 0.9 1.0 0.95 -0.05 - 25 Short Medium 1.0 0.9 0.95 1.1 0.9 1.00 0.05 - 26 Short Medium 0.7 0.8 0.75 1.0 0.9 0.95 0.20 - 27 Short Medium 1.0 1.1 1.05 1.0 1.1 1.05 0.00 - 28 Short Medium 0.9 0.7 0.80 1.0 0.9 0.95 0.15 - 29 Extended Medium 1.0 1.0 1.00 0.7 0.8 0.75 -0.25 - 30 Extended Medium 0.8 0.6 0.70 1.1 0.9 1.00 0.30 - 31 Extended Medium 0.5 0.6 0.55 0.9 0.9 0.90 0.35 - 32 Extended Medium 0.9 0.9 0.90 0.9 0.9 0.90 0.00 - 33 Extended Medium 0.8 0.8 0.80 0.6 0.7 0.65 -0.15 - 34 Extended Medium 0.5 0.5 0.50 0.8 0.9 0.85 0.35 - 35 Short High 1.1 1.0 1.05 0.9 0.9 0.90 -0.15 - 36 Short High 1.1 1.0 1.05 1.9 1.9 1.90 0.85 15** 37 Short High 1.0 1.1 1.05 0.9 0.8 0.85 -0.20 - 38 Extended High 0.8 0.9 0.85 4.2 3.2 3.70 2.85 15** 39 Extended High 0.7 0.5 0.60 2.6 2.7 2.65 2.05 15** 40 Extended High 1.0 1.0 1.00 3.5 3.0 3.25 2.25 25** * Equivocal slight swelling without obvious borders. ** Plaque-like irregular area of induration without redness, distinct borders.


21

Appendix 2: BOVIGAM, Bovine gamma interferon test results

EIA using uncentrifuged thawed plasma samples week 14

Sheep Optical densities at 450 nm control avian bovine av-con bo-con 1 2 3 4 0.293 0.303 0.310 0.010 0.017 5 0.437 0.539 0.432 0.102 -0.005 6 0.287 0.312 0.260 0.025 -0.027 7 8 9 0.311 0.365 0.347 0.054 0.036 10 0.278 0.289 0.228 0.011 -0.050 control group means 0.040 -0.006 11 12 13 14 15 16 17 0.137 0.185 0.150 0.048 0.013 18 0.400 0.465 0.714 0.065 0.314 19 0.132 0.191 0.252 0.059 0.120 20 0.290 0.311 0.338 0.021 0.048 21 0.198 0.401 0.294 0.203 0.096 22 0.253 0.285 0.251 0.032 -0.002 low group means 0.071 0.098 23 24 25 26 27 28 29 0.275 0.265 0.265 -0.010 -0.010 30 0.119 0.189 0.134 0.070 0.015 31 0.402 0.409 0.405 0.007 0.003 32 0.472 0.447 0.508 -0.025 0.036 33 0.349 0.409 0.432 0.060 0.083 34 0.352 0.973 0.730 0.621 0.378 medium group means 0.121 0.084 35 36 37 38 0.107 0.569 0.235 0.462 0.128 39 0.124 0.172 0.134 0.048 0.010 40 0.098 0.216 0.164 0.118 0.066

high group means 0.209 0.068 (OD's for at least one antigen from first EIA were in excess of 0.5) (These samples selected for retest as most likely to be true positives)


22


Repeat EIA using centrifuged thawed plasma samples week 0 week2

Sheep Optical densities at 450 nm Optical densities at 450 nm control avian bovine av-con bo-con control avian bovine av-con bo-con 1 0.036 0.036 0.043 0.000 0.007 0.030 0.048 0.045 0.018 0.015 2 0.041 0.041 0.038 0.000 -0.003 0.039 0.058 0.042 0.019 0.003 3 0.044 0.029 0.026 -0.015 -0.018 0.038 0.038 0.045 0.000 0.007 4 0.037 0.036 0.037 -0.001 0.000 0.036 0.048 0.038 0.012 0.002 5 0.047 0.069 0.062 0.022 0.015 0.032 0.063 0.062 0.031 0.030 6 0.037 0.077 0.044 0.040 0.007 0.105 0.054 0.046 -0.051 -0.059 7 0.035 0.039 0.029 0.004 -0.006 0.036 0.042 0.045 0.006 0.009 8 0.075 0.280 0.122 0.205 0.047 0.040 0.093 0.037 0.053 -0.003 9 0.045 0.051 0.036 0.006 -0.009 0.036 0.029 0.025 -0.007 -0.011 10 0.043 0.197 0.050 0.154 0.007 0.108 0.037 0.046 -0.071 -0.062

control group means 0.042 0.005 0.001 -0.007

11 0.026 0.033 0.255 0.007 0.229 0.038 0.051 0.032 0.013 -0.006 12 0.032 0.039 0.036 0.007 0.004 0.033 0.040 0.036 0.007 0.003 13 0.034 0.044 0.041 0.010 0.007 0.036 0.044 0.043 0.008 0.007 14 0.034 0.044 0.036 0.010 0.002 0.036 0.035 0.038 -0.001 0.002 15 0.029 0.031 0.035 0.002 0.006 0.046 0.050 0.045 0.004 -0.001 16 0.042 0.046 0.036 0.004 -0.006 0.033 0.071 0.046 0.038 0.013 17 0.041 0.087 0.054 0.046 0.013 0.034 0.050 0.074 0.016 0.040 18 0.035 0.038 0.033 0.003 -0.002 0.034 0.044 0.090 0.010 0.056 19 0.030 0.050 0.041 0.020 0.011 0.034 0.044 0.096 0.010 0.062 20 0.036 0.045 0.035 0.009 -0.001 0.047 0.043 0.065 -0.004 0.018 21 0.047 0.054 0.049 0.007 0.002 0.039 0.058 0.038 0.019 -0.001 22 0.033 0.037 0.092 0.004 0.059 0.037 0.058 0.036 0.021 -0.001

low group means 0.011 0.027 0.012 0.016

23 0.025 0.040 0.029 0.015 0.004 0.030 0.065 0.035 0.035 0.005 24 0.047 0.039 0.042 -0.008 -0.005 0.039 0.034 0.042 -0.005 0.003 25 0.042 0.089 0.052 0.047 0.010 0.052 0.060 0.047 0.008 -0.005 26 0.038 0.058 0.073 0.020 0.035 0.040 0.036 0.036 -0.004 -0.004 27 0.028 0.046 0.071 0.018 0.043 0.042 0.036 0.042 -0.006 0.000 28 0.038 0.241 0.129 0.203 0.091 0.039 0.111 0.038 0.072 -0.001 29 0.034 0.042 0.055 0.008 0.021 0.044 0.044 0.060 0.000 0.016 30 0.049 0.100 0.057 0.051 0.008 0.032 0.057 0.053 0.025 0.021 31 0.034 0.043 0.037 0.009 0.003 0.033 0.041 0.033 0.008 0.000 32 0.038 0.039 0.038 0.001 0.000 0.034 0.057 0.037 0.023 0.003 33 0.039 0.040 0.025 0.001 -0.014 0.036 0.045 0.049 0.009 0.013 34 0.060 0.183 0.062 0.123 0.002 0.036 0.296 0.044 0.260 0.008

medium group means 0.041 0.017 0.035 0.005

35 0.057 0.054 0.058 -0.003 0.001 0.028 0.033 0.028 0.005 0.000 36 0.051 0.135 0.039 0.084 -0.012 0.036 0.112 0.035 0.076 -0.001 37 0.039 0.048 0.026 0.009 -0.013 0.049 0.047 0.037 -0.002 -0.012 38 0.042 0.037 0.056 -0.005 0.014 0.037 0.051 0.064 0.014 0.027 39 0.042 0.091 0.055 0.049 0.013 0.026 0.054 0.040 0.028 0.014 40 0.058 0.066 0.032 0.008 -0.026 0.035 0.041 0.044 0.006 0.009

high group means 0.024 -0.004 0.021 0.006


23


Repeat EIA using centrifuged thawed plasma samples week 4 week 6 Sheep Optical densities at 450 nm Optical densities at 450 nm control avian bovine av-con bo-con control avian bovine av-con bo-con 1 0.045 0.063 0.049 0.018 0.004 0.053 0.073 0.041 0.020 -0.012 2 0.037 0.042 0.041 0.005 0.004 0.039 0.045 0.034 0.006 -0.005 3 0.028 0.028 0.032 0.000 0.004 0.056 0.047 0.304 -0.009 0.248 4 0.044 0.038 0.067 -0.006 0.023 0.041 0.051 0.064 0.010 0.023 5 0.043 0.072 0.053 0.029 0.010 0.048 0.070 0.050 0.022 0.002 6 0.030 0.095 0.054 0.065 0.024 0.036 0.084 0.034 0.048 -0.002 7 0.033 0.052 0.056 0.019 0.023 0.033 0.049 0.039 0.016 0.006 8 0.085 0.173 0.077 0.088 -0.008 0.165 0.224 0.143 0.059 -0.022 9 0.038 0.040 0.040 0.002 0.002 0.046 0.051 0.050 0.005 0.004 10 0.045 0.139 0.067 0.094 0.022 0.074 0.189 0.117 0.115 0.043 control group means 0.031 0.011 0.029 0.029 11 0.036 0.047 0.101 0.011 0.065 0.043 0.047 0.031 0.004 -0.012 12 0.108 0.042 0.032 -0.066 -0.076 0.034 0.037 0.071 0.003 0.037 13 0.029 0.034 0.030 0.005 0.001 0.037 0.058 0.056 0.021 0.019 14 0.040 0.061 0.054 0.021 0.014 0.037 0.040 0.042 0.003 0.005 15 0.039 0.057 0.044 0.018 0.005 0.029 0.075 0.055 0.046 0.026 16 0.036 0.059 0.046 0.023 0.010 0.038 0.055 0.046 0.017 0.008 17 0.065 0.055 0.052 -0.010 -0.013 0.061 0.067 0.070 0.006 0.009 18 0.037 0.035 0.071 -0.002 0.034 0.033 0.038 0.043 0.005 0.010 19 0.063 0.049 0.049 -0.014 -0.014 0.035 0.045 0.045 0.010 0.010 20 0.037 0.048 0.089 0.011 0.052 0.031 0.044 0.059 0.013 0.028 21 0.030 0.036 0.028 0.006 -0.002 0.052 0.059 0.068 0.007 0.016 22 0.035 0.036 0.038 0.001 0.003 0.034 0.033 0.033 -0.001 -0.001 low group means 0.000 0.007 0.011 0.013 23 0.040 0.105 0.043 0.065 0.003 0.034 0.057 0.050 0.023 0.016 24 0.037 0.038 0.040 0.001 0.003 0.035 0.052 0.039 0.017 0.004 25 0.032 0.043 0.041 0.011 0.009 0.046 0.045 0.044 -0.001 -0.002 26 0.034 0.042 0.037 0.008 0.003 0.039 0.039 0.053 0.000 0.014 27 0.082 0.079 0.058 -0.003 -0.024 0.037 0.038 0.039 0.001 0.002 28 0.046 0.635 0.241 0.589 0.195 0.037 0.065 0.083 0.028 0.046 29 0.030 0.034 0.033 0.004 0.003 0.037 0.033 0.046 -0.004 0.009 30 0.050 0.130 0.056 0.080 0.006 0.035 0.055 0.054 0.020 0.019 31 0.046 0.058 0.049 0.012 0.003 0.032 0.042 0.032 0.010 0.000 32 0.032 0.061 0.044 0.029 0.012 0.029 0.037 0.030 0.008 0.001 33 0.034 0.065 0.040 0.031 0.006 0.032 0.034 0.029 0.002 -0.003 34 0.044 0.469 0.043 0.425 -0.001 0.036 0.375 0.057 0.339 0.021 medium group means 0.104 0.018 0.037 0.011 35 0.052 0.057 0.059 0.005 0.007 0.035 0.036 0.034 0.001 -0.001 36 0.033 0.153 0.062 0.120 0.029 0.032 0.068 0.042 0.036 0.010 37 0.033 0.034 0.031 0.001 -0.002 0.037 0.030 0.032 -0.007 -0.005 38 0.034 0.045 0.085 0.011 0.051 0.043 0.040 0.043 -0.003 0.000 39 0.036 0.099 0.048 0.063 0.012 0.031 0.061 0.049 0.030 0.018 40 0.039 0.046 0.048 0.007 0.009 0.033 0.036 0.036 0.003 0.003 high group means 0.035 0.018 0.010 0.004


24


Repeat EIA using centrifuged thawed plasma samples week 7 week 8

Sheep Optical densities at 450 nm Optical densities at 450 nm control avian bovine av-con bo-con control avian bovine av-con bo-con 1 0.031 0.036 0.034 0.005 0.003 2 0.043 0.071 0.046 0.028 0.003 3 0.034 0.042 0.038 0.008 0.004 4 0.042 0.040 0.040 -0.002 -0.002 5 0.037 0.038 0.049 0.001 0.012 6 0.033 0.032 0.035 -0.001 0.002 7 0.041 0.126 0.083 0.085 0.042 8 0.077 0.125 0.093 0.048 0.016 9 0.036 0.039 0.039 0.003 0.003 10 0.080 0.101 0.082 0.021 0.002

control group means 0.035 0.014 0.004 0.003

11 0.046 0.063 0.039 0.017 -0.007 12 0.042 0.059 0.045 0.017 0.003 13 0.039 0.046 0.127 0.007 0.088 14 0.034 0.086 0.082 0.052 0.048 15 0.041 0.114 0.061 0.073 0.020 16 0.046 0.062 0.081 0.016 0.035 17 0.043 0.066 0.049 0.023 0.006 18 0.039 0.045 0.038 0.006 -0.001 19 0.051 0.038 0.049 -0.013 -0.002 20 0.041 0.057 0.052 0.016 0.011 21 0.040 0.073 0.053 0.033 0.013 22 0.032 0.201 0.045 0.169 0.013

low group means 0.030 0.031 0.039 0.007

23 0.070 0.107 0.092 0.037 0.022 24 0.027 0.051 0.031 0.024 0.004 25 0.044 0.063 0.049 0.019 0.005 26 0.044 0.045 0.046 0.001 0.002 27 0.061 0.101 0.068 0.040 0.007 28 0.029 0.149 0.095 0.120 0.066 29 0.035 0.048 0.048 0.013 0.013 30 0.055 0.128 0.106 0.073 0.051 31 0.042 0.083 0.043 0.041 0.001 32 0.044 0.051 0.033 0.007 -0.011 33 0.041 0.067 0.057 0.026 0.016 34 0.052 0.607 0.104 0.555 0.052


35 0.032 0.116 0.052 0.084 0.020 36 0.056 0.284 0.104 0.228 0.048 37 0.035 0.058 0.062 0.023 0.027 38 0.037 0.267 0.048 0.230 0.011 39 0.031 0.135 0.104 0.104 0.073 40 0.043 0.082 0.065 0.039 0.022

high group means 0.112 0.032 0.124 0.035


25


Repeat EIA using centrifuged thawed plasma samples week 10 week 12

Sheep Optical densities at 450 nm Optical densities at 450 nm control avian bovine av-con bo-con control avian bovine av-con bo-con 1 2 3 4 0.049 0.053 0.053 0.004 0.004 0.043 0.043 0.047 0.000 0.004 5 0.039 0.065 0.051 0.026 0.012 0.057 0.071 0.066 0.014 0.009 6 0.078 0.036 0.032 -0.042 -0.046 0.030 0.033 0.029 0.003 -0.001 7 8 9 0.198 0.069 0.044 -0.129 -0.154 0.037 0.042 0.040 0.005 0.003 10 0.077 0.184 0.105 0.107 0.028 0.036 0.033 0.036 -0.003 0.000

control group means -0.007 -0.031 0.004 0.003

11 12 13 14 15 16 17 0.045 0.144 0.084 0.099 0.039 0.055 0.093 0.068 0.038 0.013 18 0.031 0.037 0.056 0.006 0.025 0.038 0.035 0.035 -0.003 -0.003 19 0.037 0.061 0.045 0.024 0.008 0.047 0.051 0.050 0.004 0.003 20 0.042 0.057 0.076 0.015 0.034 0.040 0.049 0.061 0.009 0.021 21 0.034 0.062 0.093 0.028 0.059 0.036 0.039 0.058 0.003 0.022 22 0.055 0.041 0.036 -0.014 -0.019 0.032 0.029 0.037 -0.003 0.005

low group means 0.026 0.024 0.008 0.010

23 24 25 26 27 28 29 0.051 0.049 0.035 -0.002 -0.016 0.038 0.036 0.034 -0.002 -0.004 30 0.037 0.090 0.066 0.053 0.029 0.035 0.039 0.039 0.004 0.004 31 0.038 0.041 0.043 0.003 0.005 0.040 0.244 0.056 0.204 0.016 32 0.038 0.087 0.047 0.049 0.009 0.034 0.045 0.036 0.011 0.002 33 0.047 0.163 0.117 0.116 0.070 0.046 0.058 0.063 0.012 0.017 34 0.043 0.288 0.131 0.245 0.088 0.060 0.285 0.137 0.225 0.077


35 36 37 38 0.044 0.082 0.061 0.038 0.017 0.036 0.132 0.113 0.096 0.077 39 0.071 0.230 0.070 0.159 -0.001 0.032 0.100 0.089 0.068 0.057 40 0.038 0.128 0.063 0.090 0.025 0.038 0.141 0.132 0.103 0.094

high group means 0.096 0.014 0.089 0.076


26


Repeat EIA using centrifuged thawed plasma samples week 14 Sheep Optical densities at 450 nm control avian bovine av-con bo-con 1 2 3 4 0.052 0.046 0.054 -0.006 0.002 5 0.137 0.149 0.136 0.012 -0.001 6 0.046 0.055 0.046 0.009 0.000 7 8 9 0.049 0.079 0.070 0.030 0.021 10 0.187 0.164 0.146 -0.023 -0.041 control group means 0.004 -0.004 11 12 13 14 15 16 17 0.060 0.110 0.085 0.050 0.025 18 0.046 0.059 0.054 0.013 0.008 19 0.058 0.113 0.089 0.055 0.031 20 0.074 0.044 0.045 -0.030 -0.029 21 0.089 0.231 0.166 0.142 0.077 22 0.044 0.077 0.043 0.033 -0.001 low group means 0.044 0.019 23 24 25 26 27 28 29 0.034 0.047 0.092 0.013 0.058 30 0.076 0.069 0.063 -0.007 -0.013 31 0.047 0.049 0.068 0.002 0.021 32 0.040 0.052 0.055 0.012 0.015 33 0.103 0.062 0.076 -0.041 -0.027 34 0.051 0.320 0.221 0.269 0.170 medium group means 0.041 0.037 35 36 37 38 0.037 0.337 0.158 0.300 0.121 39 0.044 0.076 0.066 0.032 0.022 40 0.034 0.086 0.082 0.052 0.048 high group means 0.128 0.064


27

Appendix 3: PARACHEK Johne's absorbed EIA test results Week 0 Week 2 Week 4 Sheep No. OD1 OD-B2 OD OD-B OD OD-B 1 0.058 0.018 0.084 0.042 0.092 0.053 2 0.064 0.023 0.089 0.047 0.087 0.048 3 0.113 0.072 0.094 0.052 0.108 0.069 4 0.147 0.107 0.107 0.065 0.123 0.084 5 0.063 0.022 0.088 0.046 0.115 0.076 6 0.062 0.022 0.093 0.051 0.108 0.069 7 0.055 0.015 0.083 0.041 0.097 0.058 8 0.090 0.050 0.079 0.037 0.113 0.074 9 0.124 0.084 0.105 0.063 0.147 0.108 10 0.064 0.024 0.083 0.041 0.139 0.100 11 0.069 0.028 0.080 0.038 0.153 0.114 12 0.066 0.026 0.093 0.051 0.126 0.087 13 0.066 0.026 0.093 0.051 0.278 0.239 14 0.052 0.012 0.067 0.025 0.114 0.075 15 0.076 0.035 0.101 0.059 0.126 0.087 16 0.087 0.047 0.100 0.058 0.150 0.111 17 0.096 0.056 0.087 0.045 0.110 0.071 18 0.145 0.105 0.111 0.069 0.129 0.090 19 0.059 0.019 0.085 0.043 0.099 0.060 20 0.079 0.038 0.157 0.115 0.146 0.107 21 0.078 0.037 0.087 0.045 0.119 0.080 22 0.064 0.024 0.104 0.062 0.093 0.054 23 0.062 0.021 0.103 0.061 0.101 0.062 24 0.106 0.066 0.101 0.059 0.107 0.068 25 0.061 0.021 0.101 0.059 0.094 0.055 26 0.071 0.031 0.100 0.058 0.114 0.075 27 0.071 0.031 0.117 0.075 0.121 0.082 28 0.062 0.021 0.090 0.048 0.085 0.046 29 0.061 0.020 0.090 0.048 0.084 0.045 30 0.064 0.023 0.148 0.106 0.147 0.108 31 0.064 0.023 0.110 0.068 0.102 0.063 32 0.065 0.025 0.095 0.053 0.101 0.062 33 0.083 0.042 0.094 0.052 0.087 0.048 34 0.062 0.022 0.078 0.036 0.064 0.025 35 0.062 0.022 0.084 0.042 0.039 0.000 36 0.058 0.017 0.097 0.055 0.040 0.001 37 0.070 0.029 0.082 0.040 0.117 0.078 38 0.064 0.024 0.078 0.036 0.082 0.043 39 0.072 0.031 0.084 0.042 0.078 0.039 40 0.096 0.056 0.111 0.069 0.040 0.001 +ve control OD 1.133 1.092 1.064 1.022 1.029 0.990 -ve control OD 0.105 0.065 0.081 0.039 0.076 0.037 blank OD 0.041 0.042 0.039 cut-off3 0.205 0.165 0.181 0.139 0.176 0.137

1 All optical density (OD) values reported are means of duplicate readings. 2 "Corrected optical densities" (sample OD minus Blank OD). 3 This value is the negative control OD plus 0.1, as per manufacturer's recommendation. Test repeated:OD 0.063, cut-off 0.188.


28

Appendix 3: PARACHEK, Johne's absorbed EIA test results Week 6 Week 7 Week 8 Sheep No. OD1 OD-B2 OD OD-B OD OD-B 1 0.136 0.091 0.096 0.056 2 0.099 0.054 0.085 0.045 3 0.114 0.069 0.097 0.057 4 0.121 0.076 0.105 0.065 5 0.099 0.054 0.087 0.047 6 0.104 0.059 0.084 0.044 7 0.096 0.051 0.094 0.054 8 0.094 0.049 0.083 0.043 9 0.125 0.080 0.093 0.053 10 0.095 0.050 0.097 0.057 11 0.104 0.059 0.087 0.047 12 0.099 0.054 0.080 0.040 13 0.110 0.065 0.115 0.075 14 0.082 0.037 0.078 0.038 15 0.102 0.057 0.122 0.082 16 0.104 0.059 0.078 0.038 17 0.112 0.067 0.067 0.027 18 0.114 0.069 0.090 0.050 19 0.086 0.041 0.106 0.066 20 0.124 0.079 0.091 0.051 21 0.117 0.072 0.133 0.093 22 0.081 0.036 0.101 0.061 23 0.086 0.041 0.108 0.068 24 0.095 0.050 0.123 0.083 25 0.099 0.054 0.086 0.046 26 0.104 0.059 0.167 0.127 27 0.096 0.051 0.107 0.067 28 0.090 0.045 0.079 0.039 29 0.115 0.070 0.118 0.078 30 0.108 0.063 0.096 0.056 31 0.109 0.064 0.093 0.053 32 0.088 0.043 0.073 0.033 33 0.138 0.093 0.153 0.113 34 0.106 0.061 0.078 0.038 35 0.117 0.072 0.087 0.047 36 0.104 0.059 0.078 0.038 37 0.106 0.061 0.113 0.073 38 0.100 0.055 0.082 0.042 39 0.113 0.068 0.081 0.041 40 0.107 0.062 0.082 0.042 +ve control 0.984 0.939 1.172 1.132 1.172 1.132 -ve control 0.086 0.041 0.074 0.034 0.074 0.034 blank 0.045 0.040 0.040 cut-off3 0.186 0.141 0.174 0.134 0.174 0.134

1 All optical density (OD) values reported are means of duplicate readings. 2 "Corrected optical densities" (sample OD minus Blank OD). 3 This value is the negative control OD plus 0.1, as per manufacture's recommendation.


29

Appendix 3: PARACHEK, Johne's absorbed EIA test results Week 10 Week 12 Week 14 Sheep No. OD1 OD-B2 OD OD-B OD OD-B 1 2 3 4 0.107 0.067 0.086 0.046 0.119 0.080 5 0.100 0.060 0.083 0.043 0.111 0.071 6 0.105 0.065 0.102 0.062 0.122 0.083 7 8 9 0.138 0.098 0.104 0.064 0.138 0.099 10 0.098 0.058 0.087 0.047 0.117 0.077 11 12 13 14 15 16 17 0.080 0.040 0.084 0.044 0.077 0.037 18 0.097 0.057 0.080 0.040 0.096 0.057 19 0.085 0.045 0.087 0.047 0.137 0.097 20 0.102 0.062 0.172 0.132 0.094 0.055 21 0.094 0.054 0.117 0.077 0.124 0.085 22 0.086 0.046 0.101 0.061 0.120 0.081 23 24 25 26 27 28 29 0.075 0.035 0.095 0.055 0.112 0.073 30 0.089 0.049 0.138 0.098 0.105 0.065 31 0.080 0.040 0.105 0.065 0.091 0.052 32 0.069 0.029 0.081 0.041 0.126 0.087 33 0.143 0.103 0.174 0.134 0.127 0.088 34 0.083 0.043 0.040 0.000 0.120 0.081 35 36 37 38 0.081 0.041 0.043 0.003 0.115 0.075 39 0.063 0.023 0.049 0.009 0.109 0.069 40 0.106 0.066 0.043 0.003 0.252 0.212 +ve control 1.140 1.100 1.140 1.100 1.032 0.992 -ve control 0.082 0.042 0.082 0.042 0.075 0.036 blank 0.040 0.040 0.040 cut-off3 0.182 0.142 0.182 0.142 0.175 0.136

1 All optical density (OD) values reported are means of duplicate readings. 2 "Corrected optical densities" (sample OD minus Blank OD). 3 This value is the negative control OD plus 0.1, as per manufacture's recommendation. Test repeated:OD 0.194, cut-off 0.188.


30

Appendix 4: Culture results, tissues collected at necropsy

Sheep Schedule Dose

Col

on

Cae

cum

ICV

Term

inal

ileu

m

Term

inal

ileu

m

Term

inal

ileu

m

Jeju

num

+ P

P

Jeju

num

- PP

Duo

denu

m

Mes

ente

ric L

N

Mes

ente

ric L

N

Mes

ente

ric L

N

Mes

ente

ric L

N

Ret

roph

aran

geal

LN

Tons

il

1 Short Control - - - - - - - - - - - - - - - 2 Short Control - - - - - - - - - - - - - - - 3 Short Control - - - - - - - - - - - - - - - 7 Short Control - - - - - - - - - - - - - - - 8 Short Control - - - - - - - - - - - - - - - 4 Extended Control - - - - - - - - - - - - - - - 5 Extended Control - - - - - - - - - - - - - - - 6 Extended Control - - - - - - - - - - - - - - - 9 Extended Control - - - - - - - - - - - - - - - 10 Extended Control - - - - - - - - - - - - - - - 11 Short Low - - - - - - - - - - - - - - - 12 Short Low - - - - - - - - - - - - - - - 13 Short Low - - - - - - - - - - - - - - - 14 Short Low - - - - - - - - - - - - - - - 15 Short Low - - - - - - - - - - - - - - - 16 Short Low - - - - - - - - - - - - - - - 17 extended Low - - - - - - - - - - - - - - - 18 extended Low - - - - - - - - - - - - - - - 19 extended Low - - - - - - - - - - - - - - - 20 extended Low - - - - - - - - - - - - - - - 21 extended Low - - - - - - - - - - - - - - - 22 extended Low - - - - - - - - - - - - - - - 23 Short Medium - - - - - - - - - - - - - - - 24 Short Medium - - - - - - - - - - - - - - - 25 Short Medium - - - - - - - - - - - - - - - 26 Short Medium - - - - - - - - - - - - - - - 27 Short Medium - - - - - - - - - - - - - - - 28 Short Medium - - - - - - - - - - - - - - - 29 extended Medium - - - - - - - - - - - - - - - 30 extended Medium - - - - - - - - - - - - - - - 31 extended Medium - - - - - - - - - - - - - - - 32 extended Medium - - - - - - - - - - - - - - - 33 extended Medium - - - - - - - - - - - - - - - 34 extended Medium - - - - - - - - - - - - - - - 35 Short High - - - - +(5) +(5) - - - +(6) +(6) +(6) - - +(7) 36 Short High - - +(5) - - - - - - - +(6) - - - - 37 Short High - - - +(6) - - - - - - - - - - - 38 Extended High - - - - - - - - - - - - - - +(6) 39 Extended High - - +(6) +(7) - +(7) - - - +(7) +(6) - - - - 40 Extended High - - +(7) +(6) +(7) - +(7) +(6) - +(7) +(6) +(6) +(7) - - + Positive growth index observed, PCR performed on all these samples These samples were confirmed by PCR and REA to be M. avium subsp. paratuberculosis - No growth index detected ( ) Figures in brackets refer to the number of weeks for the growth index in positive samples to exceed 999. * These samples did not reach 999 even after 16 weeks incubation


31

Appendix 5: Faecal culture results

Scheduled samples cultured on day of collection Sheep Schedule Dose week 0 week 2 week 4 week 6 week 8 week 10 week 12 week 14 1 Short Control - - - - 2 Short Control - - - - 3 Short Control - - - - 7 Short Control - - - - 8 Short Control - - - - 4 Extended Control - - - - - - - - 5 Extended Control - - - - - - - - 6 Extended Control - - - - - - - - 9 Extended Control - - - - - - - - 10 Extended Control - - - - - - - - 11 Short Low - - - - 12 Short Low +* - - - 13 Short Low - - - - 14 Short Low - - - - 15 Short Low - - - - 16 Short Low - - - - 17 extended Low - - - - - - - - 18 extended Low - - - - - - - - 19 extended Low - - - - - - - - 20 extended Low - - - - - - - - 21 extended Low - - - +* - - - - 22 extended Low - - - - - - - - 23 Short Medium - - - - 24 Short Medium - - - - 25 Short Medium - - - +* 26 Short Medium - - - - 27 Short Medium +(9) - - - 28 Short Medium - - - - 29 extended Medium - - - - - - - - 30 extended Medium - - - - - - - - 31 extended Medium - - - +* - - - - 32 extended Medium - - - - - - - - 33 extended Medium - - - - - - - - 34 extended Medium - - - - - - - - 35 Short High - - - - 36 Short High - - - - 37 Short High - - - - 38 Extended High - - - - 39 Extended High - - - - 40 Extended High - - - - + Positive growth index observed, PCR performed on all these samples These samples were confirmed by PCR and REA to be M. avium subsp. paratuberculosis - No growth index detected ( ) Figures in brackets refer to the number of weeks for the growth index in positive samples to exceed 999. * These samples did not reach 999 even after 16 weeks incubation


32

Appendix 5: Faecal culture results from daily collections Week sampled 1 2 3 4 5 Days after last dose: 6 7 1 2 3 6 7 1 2 3 6 1 2 3 6 1 2 3 7 Sheep 29 extended Medium - - - - - - - - - - - - - - - - - - - 30 extended Medium - - - +(7) - - - - +(8) - - - - - - - - - - 31 extended Medium - - - - - - - - - - - - - - - - - - - 32 extended Medium - - - - - - - - - - - - - - - - - - - 33 extended Medium - - +(9) - - - - - - +(8) - - - - - - - - - 34 extended Medium - - 38 Extended High - - - - - - - - - +* - - - - - - - - 39 Extended High - - - - - - - - - - +(8) - - - - - - - 40 Extended High - - - - - - +(12) - - - - - - - - - - -

+ Positive growth index observed, PCR performed on all these samples. These samples were confirmed by PCR and REA to be M. avium subsp. Paratuberculosis. - No growth index detected. ( ) Figures in brackets refer to the number of weeks for the growth index in positive samples to exceed 999. * These samples did not reach 999 even after 16 weeks incubation.


33

Appendix 5: Faecal culture results from daily collections Week sampled 6 7 8 9 10 Days after last dose: 2 3 6 7 1 2 3 1 2 6 7 1 2 3 6 9 10 13 14 Sheep 29 extended Medium - - - - - - - - - - - - - - - - - - - 30 extended Medium - - - - - - - - - - - - - - - - - - - 31 extended Medium - - - - - - - - - - - - - - - - - - - 32 extended Medium - - - - - - - - - - - - - - - - - - - 33 extended Medium - - - - - - - - - - - - - - - - - - - 34 extended Medium 38 Extended High - - - - - - - - - - - - - - +(10) - - - - 39 Extended High +* - - - - - - - - - - - - - - - - - - 40 Extended High - - - - - - - - - - - - - - +(6) - - - -

+ Positive growth index observed, PCR performed on all these samples These samples were confirmed by PCR and REA to be M. avium subsp. Paratuberculosis. - No growth index detected. ( ) Figures in brackets refer to the number of weeks for the growth index in positive samples to exceed 999. * These samples did not reach 999 even after 16 weeks incubation.


34

Appendix 5: Faecal culture results from daily collections Week sampled 11 12 13 Days after last dose: 15 16 19 21 22 23 26 27 28 29 30 Sheep 29 extended Medium - - - - - - - - - - - 30 extended Medium - - - - - - - - - - - 31 extended Medium - - - - - - - - - - - 32 extended Medium - - - - - - - - - - - 33 extended Medium - - - - - - - - - - - 34 extended Medium 38 Extended High - - - - - - - - - - - 39 Extended High - - - - - - - - - - - 40 Extended High - - - - - - - - - - -

+ Positive growth index observed, PCR performed on all these samples. These samples were confirmed by PCR and REA to be M. avium subsp. Paratuberculosis. - No growth index detected. ( ) Figures in brackets refer to the number of weeks for the growth index in positive samples to exceed 999. * These samples did not reach 999 even after 16 weeks incubation.

pilot study: tracer weaner trial for ovine johne’s disease

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